Abstract The dramatic increase in the number of patients being diagnosed with incidental pancreatic cysts through improved imaging represents a unique opportunity to detect and treat cystic precursor lesions before the onset of malignancy. However, pancreatic cysts also represent a major clinical challenge because current diagnostics do not adequately reflect the biology of cyst malignant transformation. Misregulated pericellular proteolysis is a hallmark of invasive cancer. Therefore, we are exploiting the activities of proteases in cyst fluid to develop an enzyme-based diagnostic test for the sensitive and specific identification of pre-malignant pancreatic lesions. Our goals are to develop a rapid and minimally invasive assay that improves patient stratification over current standard diagnostic markers and guides clinical decision-making to avoid unnecessary surgical intervention. The Alaunus Biosciences diagnostic pipeline takes advantage of a substrate profiling technology developed in the Craik Laboratory at UCSF referred to as Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). The MSP-MS assay uses a peptide library platform to monitor global protease substrate specificity and kinetic efficiency in complex biological samples in an unbiased manner. Through applying this substrate profiling approach to small volumes of cyst fluid (< 1 ?L) obtained by endoscopic ultrasound, we have identified proteases that have highly increased activity in pre-malignant cysts and developed a proof-of-principle fluorogenic substrate assay that achieves improved sensitivity and specificity compared to the current clinical standard (CEA). In Aim 1, we propose to use our activity-based profiling approach to identify additional distinguishing protease markers in an expanded patient cohort (and already evident in our existing cyst fluid data). In Aim 2, substrate specificity profiles then will be used to rationally design and optimize specific fluorogenic peptide substrates that are cleaved by the target proteases discovered. Lead candidate substrates will be benchmarked in a blinded validation cohort for their performance compared to CEA in distinguishing pre-malignant from benign cysts. If successful, this proposal will lay the groundwork for an actionable diagnostic test that will enable early identification of pre-malignant cysts and transform the clinical management of these challenging lesions.